Non-infectious, replication-defective, self-assembling HIV-1 viral particles containing antigenic markers in the gag coding region

ABSTRACT

The present invention is concerned with the ability to differentiate between infection by HIV and immunization with an immunogenic preparation. Accordingly, the present invention provides a non-infectious, non-replicating, HIV retrovirus-like particle containing a heterologous antigenic marker, comprising an assembly of (a) an env gene product; (b) a pol gene product; (c) a gag gene product; and, (d) at least one non-retroviral, non-mammalian antigenic marker. The antigenic marker may have between 5 and 100 amino acid residues. In one embodiment, the amino acid sequence contains a tobacco mosaic virus (TMV) epitope obtained from the coat protein. In another embodiment, the marker is inserted into the gag gene product at an antigenically-active insertion site. The presence of the antigenic marker enables recognition that antiserum containing anti-HIV antibodies has been generated by exposure to the non-infectious retrovirus-like particles, as opposed to wild-type HIV infection, by testing for antibodies specific to the antigenic marker.

FIELD OF THE INVENTION

The present invention relates to the field of immunology and isparticularly concerned with antigenically-marked non-infectiousretrovirus-like particles (sometimes termed pseudovirions).

BACKGROUND OF THE INVENTION

Human immunodeficiency virus is a human retrovirus and is theetiological agent of acquired immunodeficiency syndrome (AIDS). SinceAIDS was first reported in the US in 1981, more than 194,000 people havedied of AIDS and over 330,000 cases of HIV infection have been reportedin the US alone. Worldwide it is estimated that more than 14 millionpeople have been infected with HIV.

More than 100 AIDS-related medicines are in human clinical trials orawaiting FDA approval but there is currently no cure for the disease.

There is therefore a clear need for immunogenic preparations useful asvaccine candidates, as antigens in diagnostic assays and kits and forthe generation of immunological reagents for diagnosis of HIV and otherretroviral disease and infection.

Particular prior art immunogenic preparations include non-infectious,non-replicating HIV-like particles. Thus PCT applications WO 93/20220published Oct. 14, 1993 and WO 91/05860 published May 2, 1990 (WhiteheadInstitute for Biomedical Research), teach constructs comprising HIVgenomes having an alteration in a nucleotide sequence which is criticalfor genomic RNA packaging, and the production of non-infectiousimmunogenic HIV particles produced by expression of these constructs inmammalian cells.

PCT application WO 91/07425 published May 30, 1991 (Oncogen LimitedPartnership) teaches non-replicating retroviral particles produced bycoexpression of mature retroviral core and envelope structural proteinssuch that the expressed retroviral proteins assemble into buddingretroviral particles. A particular non-replicating HIV-1 like particlewas made by coinfecting mammalian host cells with a recombinant vacciniavirus carrying the HIV-1 gag and protease genes and a recombinantvaccinia virus carrying the HIV-1 env gene.

In published PCT application WO 91/05864 in the name of the assigneehereof, (which is incorporated herein by reference thereto) there isdescribed particular non-infectious non-replicating retrovirus-likeparticles containing at least gag, pol and env proteins in their naturalconformation and encoded by a modified retroviral genome deficient inlong terminal repeats and containing gag, pol and env genes in theirnatural genomic arrangement.

Since there is no vaccine nor effective treatment for AIDS and sincesuch prior art HIV-like particles contain many of the HIV proteins intheir natural conformations, a host immunized therewith may mount animmune response immunologically indistinguishable from infection by HIV.Heat-inactivated anti-HIV antiserum obtained from HIV-infected peopleand inactivated HIV are currently commercially available as componentsof many diagnostic methods. For safety, ease of handling, shipping,storage and use it may be preferable to replace such heat-inactivatedantisera and antigens by non-infectious HIV and antisera generated byimmunization with non-infectious HIV particles as described above.Furthermore, antisera generated by immunization with thesenon-infectious HIV particles do not require heat inactivation to removeinfectious HIV. However, because of the seriousness of HIV infection itis desirable to be able to distinguish between inactivated HIV andnon-infectious, non-replicating HIV particles and antisera generated byvirulent HIV and non-infectious, non-replicating HIV particles. Thus, inthe development of AIDS vaccine candidates, immunogenic preparations anddiagnostic methods and kits, it would be useful to provide an HIV-likeparticle immunologically or otherwise distinguishable from virulent HIV.

SUMMARY OF THE INVENTION

The present invention is concerned with the ability to differentiatebetween infection by HIV or another retrovirus, particularly a humanretrovirus, and immunization with an immunogenic preparation. Thepresent invention is also concerned with the ability to differentiatebetween inactivated virulent HIV and non-infectious non-replicatingHIV-like particles. The present invention incorporates a marker into anon-infectious, retrovirus-like particle.

Accordingly, in one aspect, the present invention provides anon-infectious retrovirus-like particle, comprising an assembly of (a)an env gene product; (b) a pol gene product; (c) a gag gene product; and(d) at least one antigenic marker which is non-retroviral or non-HIVretroviral.

The at least one antigenic marker may have about 5 to about 100 aminoacid residues, particularly about 10 to about 75 amino acid residues.The antigenic marker may comprise at least one antigenic epitope fromanother virus. The invention is illustrated, in one embodiment, by atleast one antigenic epitope from tobacco mosaic virus (TMV) coatprotein, specifically including an amino acid sequence AFDTRNRIIEVEN(SEQ ID NO: 1) or a portion, variation or mutant thereof capable ofeliciting antibodies that recognize this sequence, or multiple copies,specifically from 1 to 4, of such amino acid sequence.

The antigenic marker may be incorporated into the assembly of env, poland gag gene products in any convenient manner. In one embodiment of theinvention, the marker sequence is contained within the gag gene productto form a hybrid gag gene product having the particle-formingcharacteristics of unmodified gag gene product. The marker sequence maybe contained within the gag gene product by insertion of the antigenicmarker into the gaq gene product at an antigenically-active insertionsite.

In one specific embodiment of the invention, the insertion site may bethat located between amino acid residues 210 and 211 of the gag geneproduct of the HIV-1 LAI isolate or the corresponding location of otherretroviral gag gene products.

The marker sequence also may be provided by deleting or preventingproduction of an amino acid sequence that corresponds to an epitope of aretroviral protein. Such epitope may comprise the immunodominant epitopeof gp41, which provides endogenous anchoring function. When suchendogenous anchoring function is removed in this way, the anchoringfunction is provided by a different antigenic anchor sequence.

Accordingly, in another aspect of the present invention, there isprovided a non-infectious retrovirus-like particle, comprising anassembly of (a) a modified env gene product in which endogenousanchoring function has been replaced by a different anchor sequenceoperatively connected to the env gene product to anchor the env geneproduct to the retrovirus-like particle; (b) a pol gene product; and (c)a gag gene product.

The anchor sequence, which may be antigenic, may have between about 5and about 100 amino acid residues, preferably about 10 to about 75 aminoacid residues. The anchor sequence may comprise at least a portion of atransmembrane component of a membrane-spanning protein, particularly aglycoprotein. Such glycoprotein may be any convenient glycoprotein, suchas an influenza virus protein, particularly a human influenza virusprotein, or an avian influenza virus protein.

The anchor sequence may include an amino acid sequenceWILWISFAISCFLLCVVLLGFIMW (SEQ ID NO: 2) or a portion, variation ormutant thereof capable of producing antibodies that recognize thatsequence, an amino acid sequence STVASSLALAIMIAGLSFWMCSNGSLQ (SEQ ID NO:3) or a portion, variation or mutant thereof capable of producingantibodies that recognize that sequence; or an amino acid sequenceWILWISFAISCFLLCVVCWGSSCGPAKKATLGATFAFDSKEEWCREKK EQWE (SEQ ID NO: 4) ora portion, variation or mutant thereof capable of producing antibodiesthat recognize that sequence.

The anchor sequence preferably is inserted into the env gene productadjacent to and upstream of functional cleavage sites of the env geneproduct. The insertion site preferably is located between amino acidresidues 507 and 508 of the env gene product of the HIV-1 LAI isolate orthe corresponding location of other retroviral env gene products.

The retrovirus-like particle preferably is one in which the env, pol andgag gene products correspond to the env, pol and gag gene products of ahuman retrovirus, particularly HIV-1, HIV-2, HTLV-1 or HTLV-2.Specifically, the human retrovirus may be HIV-1 and the env gene productmay be an LAI env gene product, an MN env gene product, an env geneproduct from a primary HIV-1 isolate, or an env gene productantigenically equivalent thereto.

The present invention also includes nucleic acid molecules encodingnon-infectious, retrovirus-like particles of the invention. Accordingly,in another aspect of the invention, there is provided a nucleic acidmolecule encoding a non-infectious retrovirus-like particle, comprisinga modified retroviral genome deficient in long terminal repeats andcontaining gag, pol and env genes in their natural genomic arrangementand a segment encoding at least one antigenic marker which isnon-retroviral or non-HIV retroviral. The nucleic acid molecule maycomprise a DNA molecule containing the characteristic genetic elementspresent in a SacI to XhoI fragment of the genome of the HIV-1_(LAI)isolate. The modified genome also may be deficient in primer bindingsite.

In one specific illustrative embodiment of this aspect of the invention,the sequence encoding the at least one antigenic marker is inserted intothe gag gene, specifically at the PstI site at nucleotide 1415 of thegag gene of HIV-1 LAI isolate or the corresponding location of otherretroviral gag genes. One specific segment comprises from 1 to 4 copiesof a DNA sequence selected from the group consisting of:

(a) 5' GCATTCGACACTAGAAATAGAATAATAGAAGTTGAAAAT 3'; (SEQ ID NO: 5);

(b) 3' CGTAAGCTGTGATCTTTATCTTATTATCTTCAACTTTTA5'; (SEQ ID NO: 6); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequence of (a) or (b).

A variety of hybridization conditions may be employed to achieve varyingdegrees of selectivity of hybridization. For a high degree ofselectivity, stringent conditions are used to form the duplexes, such aslow salt and/or high temperature conditions, such as provided by 0.02 Mto 0.15 M NaCl at temperatures of between about 50° C. to 70° C. Forsome applications, less stringent hybridization conditions are requiredsuch as 0.15 M to 0.9 M salt, at temperatures ranging from between about20° C. to 55° C. Hybridization conditions can also be rendered morestringent by the addition of increasing amounts of formamide, todestabilize the hybrid duplex.

In a yet further embodiment of the present invention, there is provideda nucleic acid molecule encoding a non-infectious retrovirus-likeparticle, comprising a modified retroviral genome deficient in longterminal repeats and containing gag, pol and env genes in their naturalgenomic arrangement with the env gene being modified to provide thereina segment encoding an antigenic anchor sequence to anchor the env geneproduct to the retrovirus-like particle, whereby the modified env geneencodes a modified env gene product in which endogenous anchoringfunction of env has been replaced by the antigenic anchor sequence.

In one specific illustrative embodiment of this aspect of the invention,the segment encoding the antigenic marker sequence is inserted into theenv gene, specifically between nucleotides 7777 and 7778 of the env geneof the HIV LAI isolate or the corresponding location of other retroviralenv genes. One specific segment encoding the anchor sequence includes aDNA sequence selected from the group consisting of:

(a) 5' TGGATCCTGTGGATTCCTTTGCCATATCATGCTTTTTGCTTTGTGTTGTTTTGCTGGGGTTCATCATGTGG 3'; (SEQ ID NO: 7);

(b) 3' ACCTAGGACACCTAAAGGAAACGGTATAGTACGAAAAACGAAACACAACAAAACGACCCCAAGTAGTACACC 5'; (SEQ ID NO: 8); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequences of (a) or (b).

Another specific segment encoding the anchor sequence includes a DNAsequence selected from the group consisting of:

(a) 5'TCAACAGTGGCAAGTTCCCTAGCACTGGCAATCATGATAGCTGGTCTATCTTTTTGGATGTGTTCCAATGGGTCATTGCAG 3' (SEQ ID NO: 9);

(b) 3' AGTTGTCACCGTTCAAGGGATCGTGACCGTTAGTACTATCGACCAGATAGAAAAACCTACACAAGGTTACCCAGTAACGTC 5' (SEQ ID NO: 10); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequences of (a) or (b). A further specific segmentencoding the anchor sequence includes a DNA sequence selected from thegroup consisting of:

(a) 5' TGGATCCTGTGGATTTCCTTTGCCATATCATGCTTTTTGCTTTGTGTTGTTTGCTGGGGTTCATCATGTGGGCCTGCCAAAAAGGCAACATTAGGTGCAACATTTGCATTTGATAGTAAAGAAGAGTGGTGCAGAGAGAAAAGAGCAGTGGGAA 3' (SEQ ID NO: 11);

(b) 3' ACCTAGGACACCTAAAGGAAACGGTATAGTACGAAAAACGAAACACAACAAACGACCCCAAGTAGTACACCCGGACGGTTTTTCCGTTGTAATCCACGTTGTAAACGTAAACTATCATTTCTTCTCACCACGTCTCTCTTTT TTCTCGTCACCCTT 5'(SEQ ID NO: 12); and

(c) DNA sequences that hybridize with (a) or (b) under stringentconditions, particularly sequences that have at least about 90% sequenceidentity with the sequence of (a) or (b).

The present invention further includes, in an additional aspect, animmunogenic composition capable of eliciting a retroviral specificimmune response and a specific immune response against a non-retroviralmarker, comprising the retrovirus-like particles or nucleic acidmolecule provided herein, and a carrier therefor. Such composition maybe formulated for mucosal or parenteral administration, by oral, anal,vaginal or intranasal routes. The immunogenic composition may compriseat least one other immunogenic or immunostimulating material,specifically an adjuvant, such as aluminum phosphate, aluminumhydroxide, Freund's incomplete adjuvant or QS21.

In a further aspect, the present invention includes a method ofimmunizing a host to produce a retroviral specific immune response and aspecific non-retroviral immune response against an antigenic marker,comprising administering to the host an immunoeffective amount of theimmunogenic composition provided herein.

The present invention also includes diagnostic procedures and kitsutilizing these materials. Specifically, in another aspect of theinvention, there is provided a method of determining the presence ofantibodies specifically reacting with retrovirus antigens in a sample,comprising the steps of (a) contacting the sample with thenon-infectious retrovirus-like particle provided herein to producecomplexes comprising the non-infectious retrovirus-like particles andany such antibodies present in the sample specifically reactivetherewith; and (b) determining production of the complexes.

In an additional aspect of the invention, there is provided a method ofdetermining the presence of retroviral antigens in a sample, comprisingthe steps of (a) immunizing a host with the immunogenic compositionprovided herein to produce retroviral antigen-specific antibodies; (b)contacting the sample with the retroviral antigen-specific antibodies toproduce complexes comprising any retrovirus antigens in the sample andthe retroviral antigen-specific antibodies; and (c) determiningproduction of the complexes.

A further aspect of the invention provides a diagnostic kit fordetecting the presence of retroviral antigens in a sample comprising (a)at least one such retroviral antigen-specific antibody provided herein;(b) means for contacting the at least one antibody with the sample toproduce a complex comprising any retroviral antigens in the sample andthe retroviral antigen-specific antibodies; and (c) means fordetermining production of the complex.

Further, in an additional aspect of the invention, there is provided amethod of identifying antiserum generated by immunization with theimmunogenic composition provided herein, comprising detecting antibodiesin the antiserum specific for the antigenic marker.

Advantages of the present invention include:

an immunogenic retrovirus-like particle comprising gag, pol and env geneproducts in their natural conformations rendered non-infectious andnon-replicating; and

an immunogenic retrovirus-like particle immunologically distinguishablefrom a virulent retrovirus.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further understood from the followingdescription with reference to the drawings in which:

FIG. 1 shows a construction scheme of a plasmid (pMTHIV-A) encoding aretrovirus-like particle in accordance with one embodiment of theinvention;

FIG. 2 shows a construction scheme of a plasmid (pMTHIVBRU) encoding aretrovirus-like particle in accordance with a further embodiment of theinvention;

FIG. 3 shows a construction scheme of a plasmid (p83-19) encoding aretrovirus-like particle in accordance with a further embodiment of theinvention;

FIG. 4 shows a construction scheme of a plasmid (pSeBS-HA2) inaccordance with an embodiment of the invention;

FIG. 5 shows a flow diagram for gene assembly-aided mutagenesis;

FIG. 6 shows a construction scheme of a plasmid (pMTHIVHA2-701) encodinga retrovirus-like particle containing an antigenic marker sequencecomprising a portion of the transmembrane component of human influenzahemagglutinin glycoprotein in accordance with a further embodiment ofthe present invention;

FIG. 7 shows a construction scheme of a plasmid (pMTHIVmHA2) encoding aretrovirus-like particle containing a non-naturally occurring marker inaccordance with a further embodiment of the invention;

FIG. 8 shows a construction scheme for a plasmid (pMTHIVMNmHA2-5)encoding a retrovirus-like particle containing a non-naturally occurringmarker in accordance with yet a further embodiment of the invention;

FIG. 9 shows details of an oligonucleotide encoding an antigenic epitopefrom tobacco mosaic virus inserted into the gag gene product of anon-infectious non-replicating retrovirus-like particle in accordancewith another embodiment of the invention;

FIG. 10 shows a construction scheme of plasmids encoding retrovirus-likeparticles having antigenic epitopes from tobacco mosaic virus;

FIG. 11A and 11B show immunoblot analysis of antigenically markedretrovirus-like particles (pseudovirions) of the present invention; and

FIG. 12 shows an immunoblot analysis of antigenically markedretrovirus-like particles to demonstrate inclusion of the antigenicmarker in the gag gene product.

GENERAL DESCRIPTION OF INVENTION

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of HIV infections, and thegeneration of immunological reagents. A further non-limiting discussionof such uses is further presented below.

Referring to FIGS. 1 and 2, there is illustrated the construction of avector pMTHIVBRU (ATCC designation 75852 containing a modifiedretroviral genome deficient in long terminal repeats, primer bindingsite and an RNA packaging sequence, and containing gag, pol and envgenes in their natural genomic arrangement. The pol gene of pMTHIVBRUhas been modified by deletion of a portion thereof to substantiallyremove the reverse transcriptase and integrase activities thereof.Furthermore, in this particular illustrated embodiment of the invention,an oligonucleotide has been inserted within the deleted pol gene tointroduce three stop codons in three different reading frames to preventremaining sequences of integrase from being translated. The gag gene ofpMTHIVBRU has also been modified to replace the two cysteine residues(Cys³⁹² and Cys³⁹⁵) in the first Cys-His box by serines.

Thus, plasmid pMTHIVBRU encodes an HIV-like particle deficient in aplurality of elements required for infectivity and/or replication of HIVbut dispensible for virus-like particle production.

Plasmid pMTHIVBRU encodes an HIV-like particle with an envelope proteincorresponding to that of the HIV-1_(LAI) isolate. Referring to FIG. 3,there is shown a plasmid p83-19 in which the LAI envelope of pMTHIVBRUhas been substantially replaced by the MN envelope sequence. Thus,plasmid p83-19 encodes an HIV-like particle deficient in a plurality ofelements required for infectivity and/or replication of HIV butdispensible for virus-like particle production, and contains as the envgene product substantially the envelope of HIV-1 isolate MN.

Referring to FIGS. 4 to 6, there is illustrated the construction of avector pMTHIVHA2-701 containing a modified HIV genome deficient in longterminal repeats, primer binding site and an RNA packaging sequence, andcontaining gag, pol and env genes in their natural genomic arrangement.The env gene in pMTHIVHA2-701 has been modified to provide therein agene encoding a different anchor sequence to anchor the env gene productto the retrovirus-like product, whereby the modified env gene encodes amodified env gene product in which endogenous anchoring function of envhas been replaced by the different anchor sequence. In retrovirus-likeparticles encoded by pMTHIVHA2-701 an immunodominant epitope of gp41(which provides endogenous anchoring function) is no longer expressed.Thus, such retrovirus-like particles are antigenically marked in anegative manner by the absence of an amino acid sequence correspondingto an epitope of a retroviral protein. The different anchor sequence mayitself be antigenic to further provide a positive non-retroviral ornon-HIV retroviral antigenic marker for the retrovirus-like particles.

In this particular illustrated embodiment of the invention, a 135-bpsequence comprising a coding DNA fragment and a stop codon from thehuman influenza virus HA2 gene was inserted between nucleotides 7777 (G)and 7778 (A) of the HIV-1_(LAI) envelope gene to prevent synthesis ofthe HIV-1_(LAI) gp41 transmembrane glycoprotein. Plasmid pMTHIVHA2-701thus encodes an HIV-like particle wherein the gp41 transmembraneglycoprotein anchoring function has been replaced by an anchor sequencefrom the human influenza virus HA2 protein and the HA2 protein furtherprovides an antigenic marker.

Referring to FIG. 7, there is illustrated plasmid pMTHIVmHA2 which issimilar to pMTHIVHA2-701 but contains as the antigenic marker sequencereplacing the endogenous anchoring function of env an amino acidsequence with no homology to known naturally occurring proteins.

Referring to FIG. 8, there is illustrated a vector pMTHIVMNmHA2-5 (ATCCdesignation 75853) containing a modified HIV genome deficient in longterminal repeats, primer binding site and an RNA packaging sequence andcontaining gag, pol and env genes in their natural genomic arrangement.The pol gene of pMTHIVMNmHA2-5 has been modified by deletion of aportion thereof to substantially remove the reverse transcriptase andintegrase activities thereof. Furthermore, an oligonucleotide wasinserted within the deleted pol gene to introduce three stop codons inthree different reading frames to prevent remaining sequences ofintegrase from being translated. The gag gene of pMTHIVMNmHA2-5 has alsobeen modified to replace the two cysteine residues in the first Cys-Hisbox of gag by serines. In pMTHIVMNmHA2-5, the endogenous anchoringfunction of env has been replaced by an amino acid sequence with noknown homology to naturally occurring proteins. HIV-like particlesproduced from Vero cells transfected with plasmid pMTHIVMNmHA2-5 werepurified and used to immunize guinea pigs. Antisera were collected andassayed by ELISA for anti-V3 (i.e. anti-envelope) antibodies andanti-mHA2 (i.e. anti-antigenic marker) antibodies as shown in Table 1.These results indicate that the env gene product is present insubstantially its native conformation and that the antigenic marker isimmunogenic.

Although particular retrovirus-like particles have been described inwhich endogenous anchoring function of env has been replaced by theantigenic anchor sequence of particular natural and unnatural proteins,it is appreciated that many variations, adaptations and modificationscan be made to the particular means by which the endogenous anchoringfunction can be replaced without departing from the essence of theinvention.

Referring to FIGS. 9 and 10, there is illustrated plasmids (pHIV-T1;pHIV-T2(ATCC Designation 75851); pHIV-T3 and pHIV-T4) containing betweenone and four copies of a DNA sequence encoding an antigenic epitope fromTMV. In the particular embodiment s shown, the TMV epitope is insertedinto the gag gene of HIV to produce a hybrid gag gene product, and theplasmids are deficient in the plurality of elements required forinfectivity and/or replication of HIV but dispensible for virus-likeparticle production as described above. Stable cell lines were producedusing plasmids pHIV-T1, pHIV-T2, pHIV-T3 and pHIV-T4 (containing 1, 2, 3and 4 copies of the antigen epitope, respectively) that producedHIV-like particles containing the antigenic marker inserted into the gagprotein. These HIV-like particles were purified and their reactivitywith anti-HIV monoclonal antibodies (FIG. 11) and anti-TMV markerantiserum (FIG. 12) determined. The results are shown in FIGS. 11 and 12and indicate that the HIV-like particles contain gp120, gp41 and p24 insubstantially their natural conformations and that the TMV marker isable to be recognized by anti-marker antibodies.

While specific embodiments of the marker sequences, which may also be ananchor sequence, are described herein, it is apparent that any otherconvenient amino acid sequence providing marker and/or anchoringfunction may be employed herein, including the absence of an amino acidsequence that corresponds to an epitope of a retroviral protein. Theamino acid sequence providing marker function may comprise anon-naturally occurring antigenic sequence which has no homology toknown proteins. An example of such sequence is the mutant HA2 sequencedescribed above. Other examples may include antigenic regions ofnon-human or non-mammalian proteins, such as non-human or non-mammalianpathogenic or comensual organisms. An example of such sequence is theTMV described above.

It is clearly apparent to one skilled in the art, that the variousembodiments of the present invention have many applications in thefields of vaccination, diagnosis, treatment of HIV infections, and thegeneration of immunological reagents. A further non-limiting discussionof such uses is further presented below.

Vaccine Preparation and Use

It has been shown that an immunogenic preparation in accordance with theinvention can elicit an immune response. One possible use of the presentinvention is, therefore, as the basis of a potential vaccine againstretroviral diseases including AIDS and AIDS-related conditions. In afurther aspect, the invention thus provides a vaccine against AIDS andAIDS-related conditions, comprising an immunogenic composition inaccordance with the invention.

Immunogenic compositions, suitable to be used as vaccines, may beprepared from non-infectious retrovirus-like particles as disclosedherein. The immunogenic composition elicits an immune response whichproduces antibodies that are antiviral. Should the vaccinated subject bechallenged by a retrovirus, such as HIV, the antibodies bind to thevirus and thereby inactivate it.

Vaccines may be prepared as injectables, as liquid solutions oremulsions. The non-infectious retrovirus-like particles may be mixedwith pharmaceutically-acceptable excipients which are compatible withthe retrovirus-like particles. Excipients may include water, saline,dextrose, glycerol, ethanol, and combinations thereof. The vaccine mayfurther contain auxiliary substances, such as wetting or emulsifyingagents, pH buffering agents, or adjuvants to enhance the effectivenessof the vaccines. Methods of achieving an adjuvant effect for the vaccineinclude the use of agents, such as aluminum hydroxide or phosphate(alum), commonly used as 0.05 to 0.1 percent solution in phosphatebuffered saline and other adjuvants, including QS21 and incompleteFreunds adjuvant. Vaccines may be administered parenterally, byinjection subcutaneously or intramuscularly. Alternatively, theimmunogenic compositions formed according to the present invention, maybe formulated and delivered in a manner to evoke an immune response atmucosal surfaces. Thus, the immunogenic composition may be administeredto mucosal surfaces by, for example, the nasal or oral (intragastric)routes. Alternatively, other modes of administration includingsuppositories and oral formulations may be desirable. For suppositories,binders and carriers may include, for example, polyalkalene glycols ortriglycerides. Oral formulations may include normally employedincipients, such as pharmaceutical grades of saccharine, cellulose andmagnesium carbonate. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained-release formulations orpowders and contain 10 to 95% of the retrovirus-like particles of theinvention.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as is therapeutically effective,protective and immunogenic. The quantity to be administered depends onthe subject to be treated, including, for example, the capacity of theindividual's immune system to synthesize antibodies, and to produce acell-mediated immune response. Precise amounts of active ingredientrequired to be administered depend on the judgment of the practitioner.However, suitable dosage ranges are readily determinable by one skilledin the art and may be of the order of micrograms of the retrovirus-likeparticles. Suitable regimes for initial administration and booster dosesare also variable, but may include an initial administration followed bysubsequent administrations. One example of an immunization schedule isat least one pre-immunization with a retrovirus-like particle, accordingto the present invention followed by at least one secondary immunizationwith a synthetic peptide described in published European PatentPublication Number 0 570 980, assigned to the assignee hereof. Thedosage of the vaccine may also depend on the route of administration andwill also vary according to the size of the host.

Nucleic acid molecules encoding the retrovirus-like particles of thepresent invention may also be used directly for immunization byadministration of the nucleic acid molecules directly, for example byinjection to a host. Processes for the direct injection of DNA into testsubjects for genetic immunization are described in, for example, Ulmeret al, 1993 (a list of references appears at the end of the disclosureand each of the listed references is incorporated by reference withoutfurther reference thereto).

Molecules in accordance with the invention may further find use in thetreatment (prophylactic or curative) of AIDS and related conditions, byacting either to displace the binding of the HIV virus to human oranimal cells or by disturbing the 3-dimensional organization of thevirus.

A further aspect of the invention thus provides a method for theprophylaxis or treatment of AIDS or related conditions, comprisingadministering an effective amount of an immunogenic composition inaccordance with the invention.

Immunoassays

The retrovirus-like particles of the present invention are useful asimmunogens, as antigens in immunoassays including enzyme-linkedimmunosorbent assays (ELISA), RIAs and other non-enzyme linked antibodybinding assays, or procedures known in the art for the detection ofanti-retroviral (for example, HIV) HIV antibodies and retroviral antigen(for example, HIV). In ELISA assays, the retrovirus-like particles areimmobilized onto a selected surface, for example a surface capable ofbinding proteins, such as the wells of a polystyrene microtitre plate.After washing to remove incompletely adsorbed retrovirus-like particles,a non-specific protein, such as a solution of bovine serum albumin (BSA)or casein, that is known to be antigenically neutral with regard to thetest sample may be bound to the selected surface. This allows forblocking of non-specific adsorption sites on the immobilizing surfaceand thus decreases the background caused by non-specific bindings ofantisera onto the surface.

The immobilizing surface is then contacted with a sample, such asclinical or biological materials to be tested, in a manner conducive toimmune complex (antigen/antibody) formation. This may include dilutingthe sample with diluents, such as solutions of BSA, bovine gammaglobulin (BGG) and/or phosphate buffered saline (PBS)/Tween. The sampleis then allowed to incubate for from about 2 to 4 hours, at temperaturessuch as of the order of about 25° to 37° C. Following incubation, thesample-contacted surface is washed to remove non-immunocomplexedmaterial. The washing procedure may include washing with a solution,such as PBS/Tween, or a borate buffer.

Following formation of specific immunocomplexes between the test sampleand the bound retrovirus-like particles, and subsequent washing, theoccurrence, and even amount, of immunocomplex formation may bedetermined by subjecting the immunocomplex to a second antibody havingspecificity for the first antibody. If the test sample is of humanorigin, the second antibody is an antibody having specificity for humanimmunoglobulins and in general IgG. To provide detecting means, thesecond antibody may have an associated activity, such as an enzymaticactivity that will generate, for example, a colour development uponincubating with an appropriate chromogenic substrate. Quantification maythen be achieved by measuring the degree of colour generation using, forexample, a visible spectra spectrophotometer.

In one diagnostic embodiment where it is desirable to identifyantibodies that recognize a plurality of HIV isolates, a plurality ofimmunologically distinct retrovirus-like particles of the presentinvention are immobilized onto the selected surface. Alternatively, whenthe anti-HIV antibodies recognize epitopes that are highly conservedamong various HIV isolates (for example, a B-cell epitope from gag orgp41 ) a single or a limited number of retrovirus-like particles may beimmobilized. In a further diagnostic embodiment where it is desirable tospecifically identify antibodies that recognize a single HIV isolate(for example, LAI, MN, SF2 or HXB2) a single particular retrovirus-likeparticle of the present invention may be immobilized. This furtherdiagnostic embodiment has particular utility in the fields of medicine,clinical trials, law and forensic science where it may be critical todetermine the particular HIV isolate that was responsible for thegeneration of an immune response including an antibody response.

In a further diagnostic embodiment, it may be desirable to specificallyidentify immunologically distinct retroviruses, for example, HIVisolates that belong to different clades. Immunologically distinct HIVisolates may include for example, LAI, MN, SF2, HXB2 or a primary HIV-1isolate. In this diagnostic embodiment, a particular retrovirus-likeparticle of the present invention is useful for generating antibodiesincluding monoclonal antibodies that specifically recognize such animmunologically distinct HIV isolate.

It is understood that a mixture of immunologically distinctretrovirus-like particles may be used either as an immunogen in, forexample, a vaccine or as a diagnostic agent. There may be circumstanceswhere a mixture of retrovirus-like particles are used to providecross-isolate protection and/or diagnosis. In this instance, the mixtureof immunogens is commonly referred to as a "cocktail" preparation.

The present invention advantageously provides retrovirus-like particlescomprising gag, pol and env gene products substantially in their naturalconformations. Such retrovirus particles will thus be recognized byconformational anti-HIV antibodies (such as anti-env antibodies) thatmay not recognize the HIV antigen in a denatured form or a syntheticpeptide corresponding to such an HIV antigen. The retrovirus-likeparticles of the invention are therefore particularly useful as antigensand as immunogens in the generation of anti-retroviral antibodies(including monoclonal antibodies) in diagnostic embodiments.

In addition, the presence of the marker generates a specific immuneresponse thereto the detection of which by the methods described aboveenables the ready distinction between immunization of a host with theimmunogenic compositions provided herein compared to material infectionby a virulent retrovirus. The ability to effect such diagnosis anddifferentiation has advantageous utility in the fields of epidemiology,clinical trials, forensic science and immunology.

Other Uses

Molecules which bind to the retrovirus-like particles on which theinvention is based, particularly antibodies, antibody-related moleculesand structural analogs thereof, are also of possible use as agents inthe treatment and diagnosis of AIDS and related conditions.

Variants of antibodies (including variants of antigen binding site),such as chimeric antibodies, humanized antibodies, veneered antibodies,and engineered antibodies that are specific for the retrovirus-likeparticles of the invention are included within the scope of theinvention.

Antibodies and other molecules which bind to the retrovirus-likeparticles of the present invention can be used for therapeutic(prophylactic and curative) and diagnostic purposes in a number ofdifferent ways, including the following:

For passive immunization by suitable administration of antibodies,possibly humanized antibodies, to HIV infected patients.

To activate, complement or mediate antibody dependent cellularcytotoxicity (ADCC) by use of antibodies of suitable subclass or isotype(possibly obtained by appropriate antibody engineering) to be capable ofperforming the desired function.

For targeted delivery of toxins or other agents, for example, by use ofimmunotoxins comprising conjugates of antibody and a cytoindiremoiety,for binding directly or indirectly to cell-surface exposed HIV proteinsof HIV-infected cells (for example, gp120).

For targeted delivery of highly immunogenic materials to the surface ofHIV-infected cells, leading to possible ablation of such cells by eitherthe humoral or cellular immune system of the host.

For detection of HIV, using a variety of immunoassay techniques.

Thus, in yet a further diagnostic embodiment, the immunogeniccompositions of the present invention (individually, or as mixturesincluding cocktail preparations) are useful for the generation of HIVantigen specific antibodies (including monoclonal antibodies) that canbe used to detect HIV or antigens, or neutralize HIV in samplesincluding biological samples.

In an alternative diagnostic embodiment, the retrovirus-like particlesof the present invention can be used to specifically stimulate HIVspecific T-cells in biological samples from, for example, HIV-infectedindividuals for diagnosis or therapy.

Biological Deposits

Certain plasmids that encode retrovirus-like particles according toaspects of the present invention that are described and referred toherein have been deposited with the American Type Culture Collection(ATCC) located at Rockville, Md. USA pursuant to the Budapest Treaty andprior to the filing of this application. Samples of the depositedplasmids will become available to the public upon grant of a patentbased upon this United States patent application. The inventiondescribed and claimed herein is not to be limited in scope by plasmidsdeposited, since the deposited embodiment is intended only as anillustration of the invention. Any equivalent or similar plasmids thatencode similar or equivalent retrovirus-like particles as described inthis application are within the scope of the invention.

Deposit Summary

    ______________________________________    Plasmid        ATCC Designation                                Date Deposited    ______________________________________    pMTHIVBRU      75852        August 4, 1994    pMTHIVMNmHA2-5 75853    pHIV-T2        75851    ______________________________________

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific Examples. These Examples are described solely for purposes ofillustration and are not intended to limit the scope of the invention.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitations.Immunological and recombinant DNA methods may not be explicitlydescribed in this disclosure but are well within the scope of thoseskilled in the art.

EXAMPLES

Methods of molecular genetics, protein biochemistry, and immunology usedbut not explicitly described in this disclosure and these EXAMPLES areamply reported in the scientific literature and are well within theability of those skilled in the art.

Example 1

This Example describes the construction of plasmid pMTHIVBRU.

Plasmid pMTHIVBRU was constructed as shown in FIGS. 1 and 2. Thisplasmid is a modification of the expression vector pMTHIVd25 describedin Rovinski et al 1992 (the literature references are identified at theend of the specification) and which contains an RNA packaging deletion,and was engineered to contain a series of mutations/deletions. Thus, aCys-His box mutation included replacements of two cysteine codons (inSEQ ID NO: 13) with two serine codons in the first Cys-His box (SEQ IDNO: 14) of the gag protein as shown in FIG. 1. This was accomplished bya PCR-based mutagenesis method. Two primers were synthesized: theupstream primer having the sequence5'-GGACTAGTACCCTTCAGGAACAAATAGGATGGATGACAAA TAATCCACCTATCCCAGTAGGAG-3'(SEQ ID NO: 15), comprising nucleotides 1,507 to 1,567 of HIV-1_(LAI),(all nucleotide numbering is according to Wain-Hobson et al., 1985) witha SpeI site at the 5'-end; and the downstream primer having the sequence5'CTCGGGCCCTGCAATTTCTGGCTATGTGCCCTTC TTTGCCACTATTGAAACTCTTAACAATC-3'(SEQ ID NO: 16), being the reverse complement of nucleotides 2,011 to1,953 with an ApaI site at the 5'-end. In the downstream primer, twoadenosine residues representing the reverse complement of nucleotides1,963 and 1,972 (Wain Hobson et al, 1985; Myers et al, 1990) werechanged to thymidine, resulting in the replacement of the two cysteinesat amino acid positions 392 and 395 of the gag gene product with twoserines (FIG. 1). These two primers were used to amplify the SpeI-ApaIDNA fragment (nucleotides 1507 to 2006) of pMTHIV (Rovinski et al, 1992)which was used as a template. The PCR-amplified SpeI-ApaI fragment waspurified by agarose gel electrophoresis and digested with restrictionenzymes SpeI and ApaI. This fragment was used to replace thecorresponding fragment in pMTHIVd25 (Rovinski et al, 1992). Theresulting plasmid was named pMTHIV-A, which contains both the RNApackaging sequence deletion and the Cys-His box mutation.

In order to delete the reverse transcriptase and integrase, two BalIrecognition sites at nucleotides 2,655 and 4,587 of HIV-1_(LAI), wereused (FIG. 2). The 1.9-kbp fragment between the two BalI sites containsDNA sequences encoding more than 95% of the reverse transcriptase andthe first 108 amino acids of the integrase. The plasmid pMTHIV-A wasdigested with BalI. After removing the 1.9-kbp BalI fragment by gelelectrophoresis, the remaining portion of the plasmid was ligated with adouble-stranded oligonucleotide: 5'-GTATAAGTGAGTAGCGGCCGCAC-3' (only onestrand is shown --SEQ ID NO: 17) which contains three stop codons inthree different reading frames to prevent the remaining sequences ofintegrase from being translated. The resulting plasmid was termedpMTHIVBRU.

Example 2

This Example describes the construction of plasmids encoding HIV-likeparticles containing antigenically marked envelope anchors.

Plasmid p83-19 was constructed from expression vector pMTHIVBRU, asshown in FIG. 3. This plasmid contains a hybrid envelope gene which wasengineered by replacing DNA encoding most of gp120_(LAI), with thecognate DNA encoding gp120_(MN). This was accomplished by replacing aKpnI/BglII DNA fragment (nucleotides 6379 to 7668) from HIV-1_(LAI),with a KpnI/BglII DNA fragment (nucleotides 6358 to 7641) fromHIV-1_(MN).

Plasmid pMTHIVHA2-701 was constructed from expression vectors pBT1(Alizon et al, 1984) and pMTHIVd25 (Rovinski et al, 1992), as shown inFIGS. 4 to 6. The pMTHIVHA2-701 vector contains a 135-bp sequencecomprising a coding DNA fragment and a stop codon from the humaninfluenza virus HA2 gene (Min Jou et al, 1980), inserted betweennucleotides 7777(G) and 7778(A) of the HIV-1_(LAI) envelope gene(Wain-Hobson et al, 1985; Myers et al, 1990). The stop codon wasinserted to prevent synthesis of the HIV-1_(LAI) gp41 transmembraneglycoprotein. A SalI (nucleotide 5821)/BamHI (nucleotide 8522) DNAfragment from pBT1 was subcloned into pSelect (Promega) to produce pSeBS(FIG. 4). The latter plasmid was used for insertion of the 135-bp by aprocedure termed herein as `gene assembly-aided mutagenesis (GAAM)`. Amutagenic primer, which was designed to contain the 135-bp sequencecomprising a coding DNA fragment from the human influenza virus HA2 gene(Min Jou et al, 1980), was assembled as shown in FIG. 5. OligonucleotideI is a 99 mer containing (from 3' to 5') 30 bases complementary tonucleotides 7748 to 7777 of HIV-1_(LAI) (Wain-Hobson et al, 1985; Myerset al, 1990) and 69 bases which are complementary to HA2 gene sequences(Min Jou et al, 1980) encoding amino acids 180 to 202 of the HA2protein. Oligonucleotide II is a 96 mer comprising (from 3' to 5') i) 60bases complementary to HA2 gene sequences which encode amino acids 203to 221 of the HA2 protein and contain the HA2 stop codon (Min Jou et al,1980), ii) 6 bases (ATCATT --SEQ ID NO: 18) defining two more stopcodons, and iii) 30 bases complementary to nucleotides 7778 to 7807 ofHIV-1_(LAI), (Wain-Hobson et al, 1985; Myers et al, 1990).Oligonucleotide III is a bridging 30mer having 15 nucleotidescomplementary to the 5'-end of oligonucleotide I and 15 nucleotidescomplementary to the 3'-end of oligonucleotide II. Ten picomoles ofoligonucleotides I and II were mixed with 20 picomoles ofoligonucleotide III and phosphorylated at 37° C. for 1.5 h in 20 μlkinase buffer (50 mM Tris-HCl, pH 7.5, 10 MM MgCl₂, 10 mm KCl, 5 MM DTT,and 0.5 MM ATP) containing 2 units of T4 polynucleotide kinase. Theoligonucleotides were annealed by heating the mixture to 95° C. for 5min and subsequently cooling it slowly to room temperature. To thismixture was added 3 μl of 10×ligase buffer (0.5 M Tris-HCl, pH 7.4, 0-1M MgCl₂, 0.1 M DTT, 10 mM Spermidine, and 1 mg/ml BSA), 3 μl of 10 mMATP, and 5 units of T4 DNA ligase, and the ligation mixture wasincubated overnight at 16° C. to complete the assembly of the mutagenicprimer (FIG. 5). This primer was used in the mutagenesis procedurewithout further purification.

Mutagenesis was performed using the Altered Sites in vitro MutagenesisSystem from Promega (Madison, Wis.). The template for mutagenesisconsisted of the pSeBS plasmid (FIG. 4) which contained the 2.7-kbpSalI/BamHI DNA fragment of the HIV-1_(LAI) envelope gene (nucleotides5821 to 8522) cloned into the pSelect phagemid vector provided in themutagenesis kit. Following the mutagenesis procedure, putative cloneswere identified by colony hybridization with a ³² P-labelledoligonucleotide III probe. Positive clones were confirmed by DNAsequencing. One of these clones, designated pSeBS-HA2, was used for theconstruction of the final vector. To this end, the modified SalI/BamHIinsert from pSeBS-HA2 was subcloned into pMTHIVd25-dSalI; the latter isa plasmid derived from pMTHIVd25 (Rovinski et al, 1992) by partialdigestion with SalI followed by Klenow treatment to eliminate the SalIsite within the plasmid backbone. The final expression construct wasdesignated pMTHIVHA2-701.

An expression vector, pMTHIVmHA2 (shown in FIG. 7) containing aheterologous DNA sequence inserted between nucleotides 7777 (G) and 7778(A) of the HIV-1_(LAI) envelope gene (Rovinski et al, 1992; Wain-Hobsonet al, 1985) was engineered as described above. In this case, a 134-bpsequence, comprising a coding DNA fragment from the human influenzavirus HA2 gene (Min Jou et al, 1990) and 68 nucleotides that, when fusedto the HA2 sequences, encodes an amino acid sequence with no homology toknown naturally occurring proteins, was inserted downstream ofnucleotide 7777 of HIV-1_(LAI) (FIG. 7). The insertion resulted in aframeshift in the translation of HIV-1_(LAI) coding sequences, and thecreation of a stop codon (TAG) to prevent synthesis of the gp41transmembrane glycoprotein of HIV-1_(LAI). The final expressionconstruct was designated pMTHIVmHA2 (FIG. 7).

Plasmid pMTHIVMNmHA2-5 was constructed from expression vectors p83-19and pMTHIVmHA2 as shown in FIG. 8. This plasmid was designed to have allof the mutations of elements required for infectivity and/or replicationof p83-19 and to contain the 134-bp insert sequence of pMTHIVmHA2 (FIG.7). To this end, p83-19 was digested with BglII (nucleotide 7,641) andXhoI (nucleotide 8,944) to remove a 1276-bp DNA fragment which wasreplaced by the cognate BglII/XhoI fragment of pMTHIVmHA2.

Example 3

This Example describes the construction of plasmids encoding HIV-likeparticles containing antigenic epitopes from TMV.

Plasmids pHIV-T1, pHIV-T2, pHIV-T3, and pHIV-T4 represent modifiedversions of the p83-19 construct in that they contain, respectively,either one, two, three, or four copies of a double-strandedoligonucleotide (FIGS. 9, 10 and 11) comprising at least one antigenicepitope (Westhof et al, 1984; Trifilleff et al, 1991) from TMV coatprotein. The construction of these four vectors is illustrated in FIGS.9 and 10. To engineer all constructs, plasmid pMTHIV-A (FIG. 1) wasfirst digested with SacII and ApaI to isolate a 1,328-bp DNA fragmentwhich was then subcloned into pBluescript (Stratagene). The recombinantplasmid was then digested with PstI which cleaves HIV-1_(LAI) DNA atnucleotide 1,415 within the gag gene. Subsequently, either one, two,three, or four copies of the double-stranded oligonucleotide shown inFIG. 9 (coding strand: SEQ ID NO: 19, complementary strand: SEQ ID NO:20, encoded amino acids: SEQ ID NO: 21) were inserted into thisrestriction site. Finally, the resulting recombinant plasmids weredigested with SacII and ApaI to release the modified insert which wasthen cloned into the cognate region of plasmid p83-19 (FIG. 10).

The expression of retrovirus-like particles containing either the mHA2epitope or various copies of the TMV epitope is depicted in FIG. 11.Vero cells were grown to 80 % confluency and transfected with 20 μg ofplasmid DNA by the transfinity (BRL) calcium phosphate procedure.Culture supernatants were analyzed for protein expression at 48 hpost-transfection. Culture media (10 ml) from cells transfected withindividual expression constructs were collected and clarified bycentrifugation at 2,000×g (sorvall RT 6000B; Dupont Company, Wilmington,Del.) for 15 min at 4° C. Retrovirus-like particles were isolated byultra-centrifugation. Pelleted particles were suspended to 40 μl of TNE,mixed with 10 μl of 5× Laemmli sample buffer and boiled for 3 min. Viralproteins were then separated by SDS PAGE and transferred to Immobilonmembranes (Millipore, Bedford, Mass.). Membranes were blocked withBLOTTO buffer (PBS containing 5% Carnation instant nonfat dry milk,0.0001% wt/vol thimerosal, and 0.01l vol/vol antifoam A emulsion) for 2h at 25° C. and then incubated with appropriate dilutions of antibodiesovernight at 4° C. Filters were then incubated with a goat anti-mouseimmunoglobulin G antibody conjugated to alkaline phosphatase (Promega,Madison, Wis.) and reacted with the alkaline phosphatase chromogenicsubstrates nitroblue tetrazolium chloride and5-bromo-4-chloro-3-indolyphosphate ρ-toluidine salt (BRL). A cocktail ofanti-gp120, anti-gp41 , and anti-p24 antibodies was used in Panel A. Amixture of anti-gp120 and anti-p24 antibodies was used in Panel B.

The results shown in FIG. 11 demonstrate that the antigenically markedHIV-like particles produce gp120, gp41 and p24 substantially in theirnatural conformations.

Example 4

This Example describes the immunogenicity and immunoreactivity ofantigenically marked HIV-like particles.

One of plasmids pHIV-T1, pHIV-T2, pHIV-T3, or .PHIV-T4 (FIG. 10) wasco-transfected with plasmid pSV2neo into Vero cells, and stable celllines were established that produce HIV-like particles. HIV-likeparticles were purified, and their reactivity to immune sera from guineapigs immunized with a peptide corresponding to the TMV marker insertedinto the gag gene product was determined by immunoblot analysis. Toobtain the immune sera, guinea pigs were immunized with 100 μg of apeptide consisting of the TMV marker conjugated to KLH and adjuvanted inFreund's complete adjuvant. All animals were boosted three times at3-week intervals with the same peptide adjuvanted in Freund's incompleteadjuvant. Immune sera were collected two weeks after the last boostershots. The results, presented in FIG. 12, illustrate the reactivity ofthe immune sera to various forms of the gag gene product present in thevarious HIV-like particles and demonstrate the antigenicity of the TMVmarker in the context of a modified HIV-1-like particle.

Plasmid pMTHIVMNmHA2-5 was co-transfected with plasmid pSV2neo into Verocells, and a stable cell line was established that produces HIV-likeparticles. HIV-like particles were then purified, and guinea pigsimmunized with 10 μg of gag p24-equivalent amounts of HIV-like particlesadjuvanted in Freund's complete adjuvant. All animals were boosted threetimes at 3-week intervals with HIV-like particles adjuvanted in Freund'sincomplete adjuvant. Two weeks after the last booster shots, immune serawere collected and assayed by ELISA for anti-V3 and anti-mHA2 markerreactivities. The results, presented in Table 1 below, indicate thatguinea pigs immunized with HIV-like particles containing the mHA2 markerproduced antibodies capable of recognizing peptides representing themHA2 marker (MHA-1) and V3 loop neutralization domains (CLTB56, CLTB71,and CLTB73). These data, therefore, demonstrate that the mHA2 marker isimmunogenic when presented in the context of an HIV-like particle andthat antibodies are also produced against the major neutralizingdeterminants of the V3 loops from different HIV isolates.

SUMMARY OF DISCLOSURE

In summary of this disclosure, the present invention provides certainnon-infectious, non-replicating, retrovirus-like particles and nucleicacid molecules encoding them as, for example, immunogenic preparationsuseful for vaccination, the generation of retroviral-specific antiseraand as antigens in diagnostic methods and kits. The retrovirus-likeparticles may have been rendered non-infectious by modifications to thepol and/or gag gene products. Particular retrovirus-like particlescontain non-retroviral antigenic markers. Modifications are possiblewithin the scope of this invention.

                                      TABLE 1    __________________________________________________________________________    The ability of retrovirus-like particles containing an antigenic marker    to generate a    retroviral-specific immune response and a marker-specific immune    response.                                               ELISA IgG TITRES.sup.1    PEPTIDE         SEQUENCE                 SPECIFICITY                                         SEQ ID NO.                                               GP542                                                   GP543                                                       GP544    __________________________________________________________________________    MHA-1         GPAKKATLGATFAFDSKEEWCREKKEQWE                                  mHA2 marker                                         22    500 5,000                                                       2,500    CLTB56         NKRKRIHIGPGRAFYTTKN      V3 (MN)                                         23    500 500 2,500    CLTB71         NTRKSIYIGPGRAFHTTGR      V3 (SF2)                                         24    500 2,500                                                       2,500    CLTB73         NTRKRIRIQRGPGRAFVTIGK    V3 (HXB2)                                         25    500 1,000                                                       2,500    Irrelevant         MKKTRFVLNSIALGLSVLSTSFVAQATLPSFVSEQNS                                  Non-HIV                                         26    100 100 100    __________________________________________________________________________     .sup.1 Each guinea pig (GP542, GP543 and GP544) was immunized as describe     in Example 4.

REFERENCES

1. Rovinski, B., Haynes, J. R., Cao, S. X., James, O., Sia, C.,Zolla-Pazner, S., Matthews, T. J. and Klein, M. (1992) J. Virol., 66,4003-4012.

2. Wain-Hobson, S., Sonigo, P., Danos, O., Col, S. and Alizon, M. (1985)Cell, 40, 9-17.

3. Myers, G., Berzofsky, J. A., Rabson, A. B., Smith, T. F. andWong-Staal, F. (ed.) (1990) Human retroviruses and AIDS. TheoreticalBiology and Biophysics, Group T-10. Los Alamos National Laboratory, LosAlamos, N. Mex.

4. Alizon, M., Sonigo, P., Barre-Sinoussi, F., Chermann, J. C.,Tiollais, P., Montagnier, L. and Wain-Hobson, S. (1984) Nature, 312,757-780.

5. Min Jou, W., Verhoeyen, M., Devos, R., Saman, E., Fang, R.,Huylebroeck, D. and Fiers, W. (1980) Cell, 19, 683-696.

6. Westhof, E., Altschuh, D., Moras, D., Bloomer, A. C., Mondragon, A.,Klug, A. and Van Regenmortel, M. H. (1984) Nature, 311, 123-126.

7. Trifilleff, E., Dubs, M. C. and Regenmertel, M. H. V. (1991) Mol.Immunol., 28, 889-896.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 26    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 13 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - Ala Phe Asp Thr Arg Asn Arg Ile Ile Glu Va - #l Glu Asn    #                10    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 24 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cy - #s Phe Leu Leu Cys Val    #                15    - Val Leu Leu Gly Phe Ile Met Trp                20    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 27 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Ser Thr Val Ala Ser Ser Leu Ala Leu Ala Il - #e Met Ile Ala Gly Leu    #                15    - Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gl - #n    #            25    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 52 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    - Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cy - #s Phe Leu Leu Cys Val    #                15    - Val Cys Trp Gly Ser Ser Cys Gly Pro Ala Ly - #s Lys Ala Thr Leu Gly    #            30    - Ala Thr Phe Ala Phe Asp Ser Lys Glu Glu Tr - #p Cys Arg Glu Lys Lys    #         45    - Glu Gln Trp Glu        50    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 39 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #    39            ATAG AATAATAGAA GTTGAAAAT    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 39 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    #    39            TATC TTATTATCTT CAACTTTTA    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 71 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    - TGGATCCTGT GGATTCCTTT GCCATATCAT GCTTTTTGCT TTGTGTTGTT TT - #GCTGGGGT      60    #       71    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 72 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    - ACCTAGGACA CCTAAAGGAA ACGGTATAGT ACGAAAAACG AAACACAACA AA - #ACGACCCC      60    #       72    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 81 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    - TCAACAGTGG CAAGTTCCCT AGCACTGGCA ATCATGATAG CTGGTCTATC TT - #TTTGGATG      60    #81                TGCA G    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 81 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    - AGTTGTCACC GTTCAAGGGA TCGTGACCGT TAGTACTATC GACCAGATAG AA - #AAACCTAC      60    #81                ACGT C    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 156 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    - TGGATCCTGT GGATTTCCTT TGCCATATCA TGCTTTTTGC TTTGTGTTGT TT - #GCTGGGGT      60    - TCATCATGTG GGCCTGCCAA AAAGGCAACA TTAGGTGCAA CATTTGCATT TG - #ATAGTAAA     120    #      156         AGAA AAAAGAGCAG TGGGAA    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 156 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    - ACCTAGGACA CCTAAAGGAA ACGGTATAGT ACGAAAAACG AAACACAACA AA - #CGACCCCA      60    - AGTAGTACAC CCGGACGGTT TTTCCGTTGT AATCCACGTT GTAAACGTAA AC - #TATCATTT     120    #      156         TCTT TTTTCTCGTC ACCCTT    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #       12    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    #       12    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 63 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    - GGACTAGTAC CCTTCAGGAA CAAATAGGAT GGATGACAAA TAATCCACCT AT - #CCCAGTAG      60    #             63    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 62 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    - CTCGGGCCCT GCAATTTCTG GCTATGTGCC CTTCTTTGCC ACTATTGAAA CT - #CTTAACAA      60    #              62    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    #                23GCCG CAC    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 6 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    #            6    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 48 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    #                48GAAA TAGAATAATA GAAGTTGAAA ATGGTGCA    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 48 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    #                48TGAT CTTTATCTTA TTATCTTCAA CTTTTACC    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 16 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    - Gly Ala Phe Asp Thr Arg Asn Arg Ile Ile Gl - #u Val Glu Asn Gly Ala    #                15    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 29 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    - Gly Pro Ala Lys Lys Ala Thr Leu Gly Ala Th - #r Phe Ala Phe Asp Ser    #                15    - Lys Glu Glu Trp Cys Arg Glu Lys Lys Glu Gl - #n Trp Glu    #            25    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 19 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    - Asn Lys Arg Lys Arg Ile His Ile Gly Pro Gl - #y Arg Ala Phe Tyr Thr    #                15    - Thr Lys Asn    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 19 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    - Asn Thr Arg Lys Ser Ile Tyr Ile Gly Pro Gl - #y Arg Ala Phe His Thr    #                15    - Thr Gly Arg    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 21 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    - Asn Thr Arg Lys Arg Ile Arg Ile Gln Arg Gl - #y Pro Gly Arg Ala Phe    #                15    - Val Thr Ile Gly Lys                20    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 37 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    - Met Lys Lys Thr Arg Phe Val Leu Asn Ser Il - #e Ala Leu Gly Leu Ser    #                15    - Val Leu Ser Thr Ser Phe Val Ala Gln Ala Th - #r Leu Pro Ser Phe Val    #            30    - Ser Glu Gln Asn Ser            35    __________________________________________________________________________

What we claim is:
 1. A non-infectious, non-replicating HIVretrovirus-like particle containing a heterologous antigenic marker,comprising an assembly of:(a) an env gene product; (b) a pol geneproduct; (c) a gag gene product; and, (d) at least one non-retroviral,non-mammalian heterologous antigenic marker,wherein said marker, whenpresented in the context of the retrovirus-like particle, is capable ofgenerating an immune response to said antigenic marker when the particleis administered to a host, said particle being encoded by a modified HIVretroviral genome deficient in long terminal repeats (LTRs) andcontaining gag, pol, and env in their natural genomic arrangement and aheterologous nucleic acid insert encoding said at least one antigenicmarker.
 2. The retrovirus-like particle of claim 1, wherein said atleast one antigenic marker has between 5 and 100 amino acid residues. 3.The retrovirus-like particle of claim 2, wherein said at least oneantigenic marker has 10 to 75 amino acid residues.
 4. Theretrovirus-like particle of claim 2 or 3, wherein the at least oneantigenic marker comprises at least one antigenic epitope from tobaccomosaic virus coat protein.
 5. The retrovirus-like particle of claim 4,wherein the at least one antigenic epitope includes an amino acidsequence AFDTRNRIIEVEN (SEQ ID No: 1) or a portion, variation or mutantthereof capable of eliciting antibodies that recognize the sequenceAFDTRNRIIEVEN (SEQ ID No: 1).
 6. The retrovirus-like particle of claim2, wherein the at least one antigenic marker is contained within the gaggene product to form a hybrid gag gene product having theparticle-forming characteristics of an unmodified gag gene product. 7.The retrovirus-like particle of claim 6, wherein said at least oneantigenic marker is inserted into the gag gene product at anantigenically-active insertion site.
 8. The retrovirus-like particle ofclaim 7, wherein said insertion site is located between amino acidresidues 210 and 211 of the gag gene product of the HIV-1 LAI isolate orthe corresponding location of other HIV gag gene products.
 9. Theretrovirus-like particle of claim 8, wherein said at least one antigenicmarker comprises from 1 to 4 tandem copies of the amino acid sequenceAFDTRNRIIEVEN (SEQ ID No: 1) or a portion, variation or mutant thereofcapable of eliciting antibodies that recognize the sequenceAFDTRNRIIEVEN (SEQ ID No: 1).
 10. The retrovirus-like particle of claim1, wherein the human retrovirus is selected from the group consisting ofHIV-1 and HIV-2.
 11. The retrovirus-like particle of claim 10, whereinthe human retrovirus is HIV-1 and the env gene product is an LAI envgene product, an MN env gene product or an env gene product from aprimary HIV-1 isolate.
 12. An immunogenic composition capable ofeliciting a retroviral specific immune response and a specific immuneresponse against a non-retroviral marker, comprising the retrovirus-likeparticle of claim 1 and a carrier therefor.
 13. The immunogeniccomposition of claim 12 formulated for mucosal or parenteraladministration.
 14. The immunogenic composition of claim 12 formulatedfor oral, anal, vaginal, or intranasal administration.
 15. Theimmunogenic composition of claim 13 further comprising at least oneother immunogenic or immunostimulating material.
 16. The composition ofclaim 15, wherein the at least one other immunostimulating material isan adjuvant.
 17. The composition of claim 16, wherein the adjuvant isselected from the group consisting of aluminum phosphate, aluminumhydroxide, Freund's incomplete adjuvant, and QS21.
 18. A method ofimmunizing a host to produce a retroviral specific immune response and aspecific immune response against the antigenic marker, comprisingadministering to the host an immunoeffective amount of the immunogeniccomposition of claim
 12. 19. A non-infectious, non-replicating HIV-1retrovirus-like particle containing a heterologous antigenic marker,comprising an assembly of:(a) an env gene product; (b) a pol geneproduct; (c) a gag gene product; and, (d) a non-retroviral,non-mammalian heterologous antigenic marker comprising from one to fourtandemly linked copies of the amino acid sequence AFDTRNRIIEVEN (SEQ IDNO: 1) inserted into the gag gene product between amino acids 210 and211 of the HIV-1 LAI isolate,wherein said marker, when presented in thecontext of the retrovirus-like particle, is capable of generating animmune response to said antigenic marker when the particle isadministered to a host, said particle being encoded by a modified HIVretroviral genome deficient in long terminal repeats (LTRs) andcontaining gag, pol, and env in their natural genomic arrangement and aheterologous nucleic acid insert encoding said antigenic marker.